Description of the Prior Art
Hypusine [N.sub..epsilon. -(4-amino-2-hydroxybutyl)lysine], an unusual naturally occurring amino acid, having the structure: ##STR2##
was first isolated from bovine brain extracts by Shiba et al. in 1971 [Biochim. Biophys. Acta., Vol. 244, pages 523-531 (1971)]. The molecule has two chiral centers, one at position 2 and one at position 9, each of which can be classified R or S by the Cahn-Ingold-Prelog method. The (2S,9R)-diastereomer (B), formed as a ##STR3##
post-translational modification of lysine, has been shown to occur on a precursor protein of the eukaryotic initiation factor "eIF-5A" (formerly called eIF-4D or IF-M.sub.2 BX; the nomenclature for initiation factors having been revised) [Cooper et al., Proc. Natl. Acad. Sci. USA, Vol. 80, pages 1854-1857 (1983); and Safer, Eur. J. Biochem., Vol. 186, pages 1-3 (1989)]. This initiation factor 5A is unique in that it is the only known cellular protein that contains the amino acid hypusine (Hpu). In the mid-1970's, eIF-5A was shown to stimulate ribosomal subunit joining and to enhance 80 S-bound Met-t-RNA reactivity with puromycin [Anderson et al., FEBS Lett., Vol. 76, pages 1-10 (1977); and Kemper et al., J. Biol. Chem., Vol. 251, pages 5551-5557 (1976)]. Later, in 1983, Cooper et al., supra, suggested that a hypusine-modified protein serves as an important initiation factor in all growing eukaryotic cells. In 1986, Park et al. [J. Biol. Chem., Vol. 261, pages 14515-14519 (1986)] isolated the eIF-5A protein from human red blood cells and elucidated the amino acid sequence surrounding the single hypusine residue, as Thr-Gly-Hpu-His-Gly-His-Ala-Lys. Furthermore, and most interesting, because of the potential application to the control of HIV replication [Bevec et al., Proc. Natl. Acad. Sci. USA, Vol. 91, pages 10829-10833 (1994); and Ruhl et al. J. Cell Biol., Vol. 123, pages 1309-1320 (1994)], the synthesis of eIF-5A analogues is of great therapeutic significance.
Since hypusine is specific to eIF-5A, antibodies derived from hypusine-containing peptides could be used to quantitate the levels of eIF-5A directly and with high specificity. Interest in developing an antibody assay of eIF-5A to investigate the physiological role of this important initiation factor prompted total synthesis of hypusine and its (2S,9R)-diastereomer [Bergeron et al., J. Org. Chem., Vol. 58, pages 6804-6806 (1993)]. The key step in the synthesis involved the N.sub..epsilon. -alkylation of N.sub..epsilon. -benzyl-N.sub..alpha. -carbobenzoxy-(L)-lysine benzyl ester with (R)- or (S)-epichlorohydrin to give the respective (2S,9R)- and (2S,9S)-chlorohydrins. Subsequent displacement of the respective chlorides by cyanide ion provided the protected hypusine skeletons. The final step, hydrogenation over PtO.sub.2 in AcOH, followed by neutralization and re-acidification, yielded the respective (2S,9S)- and (2S,9R)-hypusine dihydrochlorides. A comparison of the reported hypusine optical rotation with that of the synthetic (2S,9R)-hypusine B confirmed the stereochemical integrity of both chiral centers throughout the synthesis.
Since there exists synthetic methodology for accessing hypusine itself, it would also be desirable to have a selectively protected hypusine reagent that could be used to incorporate this unusual amino acid into selected peptides.
It is an object of the present invention to provide such novel hypusine reagents, as well as methods for their synthesis.